The dynamics of regulatory T cells in the course of Trypanosoma cruzi infection is still debated. We previously demonstrated that acute murine T. cruzi infection results in an impaired peripheral CD4+Foxp3+ T cell differentiation due to the acquisition of an abnormal Th1-like phenotype and altered functional features, negatively impacting on the course of infection. Moreover, T. cruzi infection induces an intense thymic atrophy. As known, the thymus is the primary lymphoid organ in which thymic-derived regulatory T cells, known as tTregs, differentiate. Considering the lack of available data about the effect of T. cruzi infection upon tTregs, we examined tTreg dynamics during the course of disease. We confirmed that T. cruzi infection induces a marked loss of tTreg cell number associated to cell precursor exhaustion, partially avoided by glucocorticoid ablation- and IL-2 survival factor depletion. At the same time, tTregs accumulate within the CD4 single-positive compartment, exhibiting an increased Ki-67/Annexin V ratio compared to controls. Moreover, tTregs enhance after the infection the expression of signature markers (CD25, CD62L and GITR) and they also display alterations in the expression of migration-associated molecules (α chains of VLAs and chemokine receptors) such as functional fibronectin-driven migratory disturbance. Taken together, we provide data demonstrating profound alterations in tTreg compartment during acute murine T. cruzi infection, denoting that their homeostasis is significantly affected. The evident loss of tTreg cell number may compromise the composition of tTreg peripheral pool, and such sustained alteration over time may be partially related to the immune dysregulation observed in the chronic phase of the disease.

Author Summary

Regulatory T cells (Tregs) play a key role in balancing protective immunity and pathogenesis during diverse parasitic infections. In the context of Trypanosoma cruzi infection, some findings showed that peripheral Tregs (pTregs) might have an important role as contraregulatory mechanism, limiting tissue damage and avoiding the more severe clinical forms of chronic Chagas disease. Furthermore, there are finding showing that murine lethal T. cruzi infection causes a severe depletion of pTregs and the induction of T-bet and IFN-γ expression in the remaining pTregs, which might favours immunopathology. The thymus is a central organ of the immune system that sustains thymic Tregs (tTregs) development, but which is also a target of T. cruzi infection. We previously showed profound alterations of CD4+CD8+ double positive and CD4−CD8−double negative populations during infection. The present study confirms that T. cruzi also severely influences the thymic compartment of tTreg cells, resulting in a series of phenotypic, locational and functional disturbances. These findings showed that a parasitic infection could alter the normal homeostasis of tTregs, while in the context of T. cruzi infection these data indicate that tTregs disturbances may influence the development of chronic pathology, considering the suspected autoimmune basis of chagasic cardiomyopathy.

Oral transmission of Chagas disease has been documented in Latin American countries. Nevertheless, significant studies on the pathophysiology of this form of infection are largely lacking. The few studies investigating oral route infection disregard that inoculation in the oral cavity (Oral infection, OI) or by gavage (Gastrointestinal infection, GI) represent different infection routes, yet both show clear-cut parasitemia and heart parasitism during the acute infection. Herein, BALB/c mice were subjected to acute OI or GI infection using 5x104 culture-derived Trypanosoma cruzi trypomastigotes. OI mice displayed higher parasitemia and mortality rates than their GI counterparts. Heart histopathology showed larger areas of infiltration in the GI mice, whereas liver lesions were more severe in the OI animals, accompanied by higher Alanine Transaminase and Aspartate Transaminase serum contents. A differential cytokine pattern was also observed because OI mice presented higher pro-inflammatory cytokine (IFN-γ, TNF) serum levels than GI animals. Real-time PCR confirmed a higher TNF, IFN-γ, as well as IL-10 expression in the cardiac tissue from the OI group compared with GI. Conversely, TGF-β and IL-17 serum levels were greater in the GI animals. Immunolabeling revealed macrophages as the main tissue source of TNF in infected mice. The high mortality rate observed in the OI mice paralleled the TNF serum rise, with its inhibition by an anti-TNF treatment. Moreover, differences in susceptibility between GI
versus
OI mice were more clearly related to the host response than to the effect of gastric pH on parasites, since infection in magnesium hydroxide-treated mice showed similar results. Overall, the present study provides conclusive evidence that the initial site of parasite entrance critically affects host immune response and disease outcome. In light of the occurrence of oral Chagas disease outbreaks, our results raise important implications in terms of the current view of the natural disease course and host-parasite relationship.

Author Summary

Chagas disease caused by the protozoan Trypanosoma cruzi is endemic in Latin America and a neglected tropical disease, which affects 6–7 million people worldwide. Currently, oral transmission is the most frequent pathway of infection in Brazil but also occurs in other endemic countries. This important infection route is underestimated and understudied. Here, we demonstrate that the site of parasite entrance, in the oral cavity (OI), as observed in natural infection, or directly to the gastrointestinal tract (GI), differentially affects the host-immune response and mortality. OI promotes a severe acute disease, elevated parasitemia and TNF mediated mortality. OI showed intense hepatitis and mild heart damage. Interestingly, GI mice presented mild disease, along with less circulating TNF and higher TGF-β and IL-17 serum contents. GI animals showed mild liver damage and intense heart inflammation. Our study is a pioneer work that analyzes the features of two distinct routes of oral infection. In addition, it provides new clues for Chagas pathology and stimulates background for the elucidation of disease features in orally exposed populations.

Worldwide, around 14 million individuals are coinfected with both tuberculosis (TB) and human immunodeficiency virus (HIV). In coinfected individuals, both pathogens weaken immunological system synergistically through mechanisms that are not fully understood. During both HIV and TB infections, there is a chronic state of inflammation associated to dramatic changes in immune cytokine and endocrine hormone levels. Despite this, the relevance of immunoendocrine interaction on both the orchestration of an effective immune response against both pathogens and the control of the chronic inflammation induced during HIV, TB, or both infections is still controversial. The present study reviews immunoendocrine interactions occurring during HIV and TB infections. We also expose our own findings on immunoendocrine cross talk in HIV-TB coinfection. Finally, we evaluate the use of adrenal hormones and their derivatives in immune-therapy and discuss the use of some of these compounds like the adjuvant for the prevention and treatment of TB in HIV patients.

The protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironmental and lymphoid compartments. Acute infection results in severe atrophy of the organ and early release of immature thymocytes into the periphery. To date, the pathophysiological effects of thymic changes promoted by parasite-inducing premature release of thymocytes to the periphery has remained elusive. Herein, we show that sphingosine-1-phosphate (S1P), a potent mediator of T cell chemotaxis, plays a role in the exit of immature double-negative thymocytes in experimental Chagas disease. In thymuses from T. cruzi-infected mice we detected reduced transcription of the S1P kinase 1 and 2 genes related to S1P biosynthesis, together with increased transcription of the SGPL1 sphingosine-1-lyase gene, whose product inactivates S1P. These changes were associated with reduced intrathymic levels of S1P kinase activity. Interestingly, double-negative thymocytes from infected animals expressed high levels of the S1P receptor during infection, and migrated to lower levels of S1P. Moreover, during T. cruzi infection, this thymocyte subset expresses high levels of IL-17 and TNF-α cytokines upon polyclonal stimulation. In vivo treatment with the S1P receptor antagonist FTY720 resulted in recovery the numbers of double-negative thymocytes in infected thymuses to physiological levels. Finally, we showed increased numbers of double-negative T cells in the peripheral blood in severe cardiac forms of human Chagas disease.

Author Summary

The formation of mature lineage-committed T cells requires the specialized environment of the thymus, a central organ of the immune system supporting the development of self-tolerant T cells. Key events of intrathymic T-cell development include lineage commitment, selection events and thymic emigration. This organ undergoes physiological involution during aging. However, acute thymic atrophy can occur in the presence autoimmune diseases, malignant tumors and infections caused by intracellular pathogens. The present study shows that the protozoan parasite Trypanosoma cruzi changes the thymic microenvironmental and lymphoid compartments, resulting in premature release of very immature CD4−CD8− double-negative thymocytes, TCRneg/low, which bear a pro-inflammatory activation profile. Strikingly, we also found elevated levels of these undifferentiated T lymphocytes in the peripheral blood of patients in severe cardiac forms of chronic Chagas disease. Importantly, we provided evidence that migration of CD4−CD8− T cells from infected mouse thymus is due to sphingosine-1-phosphate receptor-1-dependent chemotaxis. These findings point to an important role for bioactive signaling sphingolipids in the thymic escape of immature thymocytes to the periphery in Chagas disease.

The relation between men and women suffering pulmonary tuberculosis is 7/3 in favor to males. Sex hormones could be a significant factor for this difference, considering that testosterone impairs macrophage activation and pro-inflammatory cytokines production, while estrogens are proinflammatory mediator’s inducer. The aim of this work was to compare the evolution of tuberculosis in male and female mice using a model of progressive disease. BALB/c mice, male and female were randomized into two groups: castrated or sham-operated, and infected by the intratracheal route with a high dose of Mycobacterium tuberculosis strain H37Rv. Mice were euthanized at different time points and in their lungs were determined bacilli loads, inflammation, cytokines expression, survival and testosterone levels in serum. Non-castrated male mice showed significant higher mortality and bacilli burdens during late disease than female and castrated male animals. Compared to males, females and castrated males exhibited significant higher inflammation in all lung compartments, earlier formation of granulomas and pneumonia, while between castrated and non-castrated females there were not significant differences. Females and castrated males expressed significant higher TNF-α, IFN γ, IL12, iNOS and IL17 than non-castrated males during the first month of infection. Serum Testosterone of males showed higher concentration during late infection. Orchidectomy at day 60 post-infection produced a significant decrease of bacilli burdens in coexistence with higher expression of TNFα, IL-12 and IFNγ. Thus, male mice are more susceptible to tuberculosis than females and this was prevented by castration suggesting that testosterone could be a tuberculosis susceptibility factor.

Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1−/−) mice have a dysregulated synthesis of glucocorticoids (GCs) during Trypanosoma cruzi acute infection. Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1−/− mice undergoing acute infection (Tc-WT and Tc-TNF-R1−/− groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1−/− compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1−/− mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1−/− mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1−/− mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.

We investigated the relationship between potentially pathogenic antibodies against a Trypanosoma cruzi ribosomal protein (P2β) and the evolution of Chagas disease and the effect of trypanocidal treatment on these variables. Seventy-eight patients with chronic Chagas disease who were followed-up for more than 20 years were divided into three groups: 30 asymptomatic persons undergoing specific treatment (group A), 37 asymptomatic persons not undergoing specific treatment (group B), and 11 patients with chronic chagasic cardiomyopathy (CCC) who were not treated. Five patients in group B showed evolution to myocardial abnormalities. Among persons with CCC, six showed no changes; the remaining persons showed progression of cardiac involvement. Levels of antibodies to P2β in persons in group A decreased from their initial values. This finding was not observed in persons in groups B and C. Comparisons at the end of the follow-up showed lower amounts of antibodies to P2β in groups A and C. These findings support the benefits of specific treatment during chronic infection.

Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in Trypanosoma cruzi (T. cruzi) acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4+CD8+ T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4+CD8+. Infected animals also exhibit enhanced levels of expression of both mRNA TNF-α receptors in the CD4+CD8+ subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event in health and disease, and that TNF-α is a further player in the process.

Tuberculosis (TB) remains the most frequent cause of illness and death from an infectious agent, and its interaction with HIV has devastating effects. We determined plasma levels of dehydroepiandrosterone (DHEA), its circulating form DHEA-suphate (DHEA-s) and cortisol in different stages of M. tuberculosis infection, and explored their role on the Th1 and Treg populations during different scenarios of HIV-TB coinfection, including the immune reconstitution inflammatory syndrome (IRIS), a condition related to antiretroviral treatment. DHEA levels were diminished in HIV-TB and HIV-TB IRIS patients compared to healthy donors (HD), HIV+ individuals and HIV+ individuals with latent TB (HIV-LTB), whereas dehydroepiandrosterone sulfate (DHEA-s) levels were markedly diminished in HIV-TB IRIS individuals. HIV-TB and IRIS patients presented a cortisol/DHEA ratio significantly higher than HIV+, HIV-LTB and HD individuals. A positive correlation was observed between DHEA-s and CD4 count among HIV-TB individuals. Conversely, cortisol plasma level inversely correlated with CD4 count within HIV-TB individuals. M. tuberculosis-specific Th1 lymphocyte count was increased after culturing PBMC from HIV-TB individuals in presence of DHEA. We observed an inverse correlation between DHEA-s plasma level and Treg frequency in co-infected individuals, and CD4+FoxP3+ Treg frequency was increased in HIV-TB and IRIS patients compared to other groups. Strikingly, we observed a prominent CD4+CD25-FoxP3+ population across HIV-TB and HIV-TB IRIS patients, which frequency correlated with DHEA plasma level. Finally, DHEA treatment negatively regulated FoxP3 expression without altering Treg frequency in co-infected patients. These data suggest an enhancing role for DHEA in the immune response against M. tuberculosis during HIV-TB coinfection and IRIS.

Genotyping studies show a polarized geographic distribution of Trypanosoma cruzi lineages in humans. Here, we assessed their distribution along Latin America through an immunological approach we designated Western blot (WB) assay with Trypomastigote small-surface antigen (TSSA) I and TSSA II (TSSA-WB). These antigens are expressed by T. cruzi I (TCI; now TcI) and T. cruzi II (TCII; reclassified as TcII to TcVI) parasites. TSSA-WB showed good concordance with genotyping tests. An unexpected frequency of TSSA II recognition was observed in Colombia, Venezuela, and Mexico (northern region of Latin America). In Argentina and Paraguay (southern region), immunophenotyping confirmed the already reported TCII (TcII to TcVI) dominance. The lineage distribution between these regions showed significant difference but not among countries within them (except for Colombia and Venezuela). TSSA-WB shows TCII emergence in the northern region where TCI was reported as dominant or even as the unique T. cruzi lineage infecting humans.

Our study investigated the circulating levels of factors involved in immune-inflammatory-endocrine-metabolic responses in patients with tuberculosis with the aim of uncovering a relation between certain immune and hormonal patterns, their clinical status and in vitro immune response. The concentration of leptin, adiponectin, IL-6, IL-1β, ghrelin, C-reactive protein (CRP), cortisol and dehydroepiandrosterone (DHEA), and the in vitro immune response (lymphoproliferation and IFN-γ production) was evaluated in 53 patients with active untreated tuberculosis, 27 household contacts and 25 healthy controls, without significant age- or sex-related differences. Patients had a lower body mass index (BMI), reduced levels of leptin and DHEA, and increased concentrations of CRP, IL-6, cortisol, IL-1β and nearly significant adiponectin values than household contacts and controls. Within tuberculosis patients the BMI and leptin levels were positively correlated and decreased with increasing disease severity, whereas higher concentrations of IL-6, CRP, IL-1β, cortisol, and ghrelin were seen in cases with moderate to severe tuberculosis. Household contacts had lower DHEA and higher IL-6 levels than controls. Group classification by means of discriminant analysis and the k-nearest neighbor method showed that tuberculosis patients were clearly different from the other groups, having higher levels of CRP and lower DHEA concentration and BMI. Furthermore, plasma leptin levels were positively associated with the basal in vitro IFN-γ production and the ConA-driven proliferation of cells from tuberculosis patients. Present alterations in the communication between the neuro-endocrine and immune systems in tuberculosis may contribute to disease worsening.

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease.

Author Summary

The thymus is a primary lymphoid organ that plays an important role on the development of the immune system and maturation of the T cell repertoire. During the normal life span, this organ undergoes involution during the aging and also in the presence of a wide variety of infectious diseases. It has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment. In the acute phase, this results in a severe atrophy of the organ and early release of immature double-positive (DP) T cells into the periphery. The effect of the changes promoted by the parasite infection on thymic central tolerance has remained not clear. The present study shows that the intrathymic key elements that promote the negative selection of thymocytes during the thymopoiesis remains functional in the acute chagasic thymic atrophy. However, we found that the DP cells released into the periphery acquire an activated phenotype and its high frequency in the peripheral blood are associated with severe cardiac forms of human chronic Chagas disease.